PROJECT SUMMARY- PROJECT 1 Cancer cells display uncontrolled inheritance of chromosomes, including errors in DNA replication and mitosis. These errors cause and propagate mutations and chromosomal abnormalities that further enhance cancer. Project 1 has been a leader in studying the mechanisms and control of inheritance of the human genome and has identified many of the key proteins that are involved in DNA synthesis at the replication fork and other proteins that are involved in the initiation of DNA replication. In the proposed studies, Project 1 will continue focusing on the mechanism and regulation of the initiation of DNA replication. Project 1 has discovered that certain initiation proteins are involved in many aspects of the cell division cycle, including centrosome duplication, centromere function and cytokinesis. Recent results also show that some initiation proteins are intimately involved in the fundamental decision of whether newly born cells will commit to a new round of cell division or enter into a period of quiescence. The proposed research will fall into three areas. Specific Aim 1 will focus on the role of the Origin Recognition Complex (ORC) subunit ORC1 and its related protein CDC6 in regulation of the commitment to cell division by controlling the expression of E2F1-regulated genes, in addition to the gene encoding Cyclin E, in cooperation with the tumor suppressor protein RB and the histone methyltransferase SUV39H1. Aim 1 will also focus on how CDC6 cooperates with ORC, RB, SUV39H1 and Cyclin dependent kinases to promote initiation of DNA replication. Specific Aim 2 will continue to study the role of the ORC subunits ORC2 and ORC3 at centromeres. ORC2 interacts with the Spindle Assembly Checkpoint protein BUBR1 only when it is phosphorylated during mitosis and defects in the BUBR1 binding domain of ORC2 cause the persistence of chromosomes that fail to align at the metaphase plate. Aim 2 will determine how ORC2 binds to BUBR1 and controls access of BUBR1. How ORC2 and ORC3 are recruited to centromeres, and the role of ORC3 interaction with the HP1 heterochromatin protein in chromosome segregation will also be studied. Project 3 will also study E2f1-regulated control of cell proliferation in a subset of breast and colon epithelial cancers have an acquired dependence of the DEAD-box RNA helicase DDX5. DDX5 is also required for progression of Acute Myeloid Leukemia (AML). This Aim will investigate how DDX5 becomes essential in some adult cancers and AML, while dispensable in normal epithelial and normal hematopoietic cells. Both RNA and protein binding partners will be identified, comparing both DDX5-dependent (DDX5-D) and DDX5-independent (DDX5-I) breast cancer cells. Exploiting the differential dependence on DDX5, Anti-Sense Oligonucleotides (ASO) will be developed for studying the role of DDX5 in mouse models for breast cancer and AML.